Bioassay and Characterization of Several Palmer Amaranth (<i>Amaranthus palmeri</i>) Biotypes with Varying Tolerances to Glyphosate

The wide distribution of Palmer amaranth (Amaranthus palmeri) in the southern US became a serious weed control problem prior to the extensive use of glyphosate-resistant crops. Currently glyphosate-resistant populations of Palmer amaranth occur in many areas of this geographic region creating an even more serious threat to crop production. Investigations were undertaken using four biotypes (one glyphosate-sensitive, one resistant from Georgia and two of unknown tolerance from Mississippi) of Palmer amaranth to assess bioassay techniques for the rapid detection and level of resistance in populations of this weed. These plants were characterized with respect to chlorophyll, betalain, and protein levels and immunological responses to an antibody of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) the target site of glyphosate. Only slight differences were found in four biotypes grown under greenhouse conditions regarding extractable soluble protein and chlorophyll content, but one biotype was found to be devoid of the red pigment, betalain. Measurement of early growth (seedling shoot elongation) of seedlings was a useful detection tool to determine glyphosate resistance. A leaf disc bioassay (using visual ratings and/or chlorophyll analysis) and an assay for shikimate accumulation were effective methods for determining herbicide resistance levels. The two unknown biotypes were found to be resistant to this herbicide. Some differences were found in the protein profiles of the biotypes, and western blots demonstrated a weak labeling of antibody in the glyphosate-sensitive biotype, whereas strong labeling occurred in the resistant plants. This latter point supports research by others, that increased copy number of the EPSPS gene (and increased EPSPS protein levels) is the resistance mechanism in this species. Results indicate the utility of certain bioassays for the determination of resistance and provide useful comparative information on the levels of inherent constituents among closely related plants.

Susceptible and Glyphosate-Resistant Palmer Amaranth (<i>Amaranthus palmeri</i>) Response to Glyphosate Using C¹⁴as a Tracer: Retention, Uptake, and Translocation

The foliar retention, absorption, translocation, and diffusion of glyphosate in glyphosate resistant-(R) and susceptible (S)-Palmer amaranth populations from seed collected in Georgia in 2007 were examined. The R population of Palmer amaranth had an elevated copy number of the EPSPS gene conferring the mechanism of resistance. When applications of 14C-glyphosate to a single leaf followed entire plant treatment with glyphosate, the distribution percentages were similar for R and S for the above and below treated leaves when harvested at 1, 6, 12, 24, and 48 hours after treatment (HAT). There were initially no differences between R and S at 1 HAT with an average of 8% absorption for both biotypes. However, data indicated that glyphosate absorption increased for R-Palmer amaranth reaching 41% within 6 HAT and was significantly different (P = 0.01) from the 28% absorbed by S-Palmer amaranth. Glyphosate resistant and susceptible Palmer amaranth averaged 44% 14C-glyphosate absorption by 24 HAT. There were no differences for 14C-glyphosate Bq/mg of plant tissue between R and S for the above the treated leaf and below the treated leaf portions of plants at 1, 6, 12, 24, or 48 HAT. However, root accumulation of 14C-glyphosate in plant tissue was significantly greater by 12 HAT for the roots of R (1.21 Bq/mg) than for S (0.51 Bq/mg). The treated leaf of the R-Palmer amaranth plants exhibited greater translocation of 14C-glyphosate in Bq/mg of tissue than the susceptible over time, indicating no detrimental effect or cost of fitness due to EPSPS gene amplification. Additionally, there were no differences in glyphosate retention in leaf discs assays between R and S biotypes. In spite of an average of 6.5 Bq efflux out of R and S leaf discs after 15 minute, only 0.4 Bq was retained after 150 minutes. Glyphosate was not retained over time in the leaf discs for R and S, and there were no biotype differences within bathing times. However, the rate of efflux (the slope of the curves) was greater for the R biotype. These data support the reported gene amplification non-target site glyphosate resistance mechanism in Palmer amaranth.

Target Site-Based Resistance to ALS Inhibitors, Glyphosate, and PPO Inhibitors in an <i>Amaranthus palmeri</i>Accession from Mississippi

Extensive acceptance of glyphosate-resistant (GR) row crops coupled with the simultaneous increase in glyphosate usage has sped the evolution of glyphosate resistance in economically important weeds. GR Amaranthus palmeri populations are widespread across the state with some exhibiting multiple resistance to acetolactate synthase (ALS) inhibiting herbicides such as pyrithiobac. A GR and ALS inhibitor-resistant accession was also resistant to the protoporphyrinogen oxidase (PPO) inhibiting herbicide fomesafen. The PPO inhibitor resistance profile and multiple herbicide resistance mechanisms in this accession were investigated. In addition to fomesafen, resistance to postemergence applications of acifluorfen, lactofen, carfentrazone, and sulfentrazone was confirmed. There was no resistance to preemergence application of fomesafen, flumioxazin, or oxyfluorfen. Molecular analysis of the ALS gene indicated the presence of point mutations leading to single nucleotide substitutions at codons 197, 377, 574, and 653, resulting in proline-to-serine, arginine-to-glutamine, tryptophan-to-leucine, and serine-to-asparagine replacements, respectively. The resistant accession contained up to 87-fold more copies of the EPSPS gene compared to a susceptible accession. A mutation leading to a deletion of glycine at codon 210 (ΔG210) of PPO2 gene was also detected. These results indicate that the mechanism of resistance in the Palmer amaranth accession is target-site based, i.e., altered target site for ALS and PPO inhibitor resistance and gene amplification for glyphosate resistance.

基于4种生态位模型的长芒苋潜在适生区预测

恶性入侵杂草长芒苋Amaranthus palmeri S.Watson对农业生产和生物多样性造成严重威胁。预测其潜在适生区对粮食安全和生物多样性保护至关重要。不同模型由于算法不同对长芒苋潜在适生区的预测结果存在差异。本研究综合4种生态位模型(MaxEnt、GARP、BIOCLIM和DOMAIN)预测长芒苋在我国的潜在适生区以提高预测准确性。结果表明,4种模型的平均AUC值均大于0.85。MaxEnt和DOMAIN的平均Kappa值大于0.81;BIOCLIM和GARP的平均Kappa值大于0.69。MaxEnt的预测精度和稳定性要略胜一筹。BIOCLIM和MaxEnt的预测结果较为收敛,DOMAIN和GARP预测的适生区范围较广,由此预测的长芒苋潜在适生区分别占我国陆地总面积的20.66%和32.38%,48.39%和49.76%。综合预测结果,长芒苋在我国的适生区主要集中在中东部地区,西北和东北地区则是长芒苋存在的边缘环境地区。

长芒苋入侵对弃耕地土壤种子库群落特征的影响

为阐明外来入侵植物长芒苋(Amaranthus palmeri)对土壤种子库群落特征的影响,选取长芒苋不同入侵压力下的弃耕地土壤种子库为研究对象,通过野外调查和萌发试验,揭示弃耕地土壤种子库群落对长芒苋入侵的响应,旨在为入侵植物的科学防控提供依据。结果表明:(1)土壤种子库共计出现30种植物,隶属于17科30属,以一年生草本为主。与轻度入侵区组相比,重度入侵区组土壤种子库物种多样性指数和种子密度都显著降低;土壤种子库物种多样性指数对土层深度的响应不明显,且入侵压力与土层深度之间无交互作用。(2)土壤种子库NMDS排序及PERMANOVA分析结果进一步表明,轻度入侵区组与重度入侵区组群落结构差异显著;重度入侵区组长芒苋已在该地建立长久性土壤种子库,影响本地生态系统的稳定性。(3)种间相关分析结果表明,轻度入侵压力和重度入侵压力均会对大豆(Glycine max)、狗尾草(Setaria viridis)和碱蓬(Suaeda glauca)3种植物产生负面效应。

ITS序列及其SNP位点在外来入侵杂草长芒苋、西部苋和糙果苋物种鉴定中的应用

对于以种子形态为物种主要识别依据的口岸部门,外来入侵杂草长芒苋、西部苋和糙果苋一直是鉴定的难点。本文对34种苋属植物的ITS序列和26SrDNA进行分析,通过SNP变异位点及特异性引物,借助PCR-RFLP方法,对长芒苋、西部苋与糙果苋进行快速、准确的分类鉴定。ITS序列分析表明,长芒苋种内变异小,种间变异显著,可与其他苋属植物明显区分。西部苋与糙果苋之间ITS序列差异小,需依据SNP位点来区别。

长芒苋——中国苋属一新归化种

首次记载长芒苋AmaranthuspalmeriS .Watson ,一种原产美国西南部和墨西哥北部的有毒植物 。

Interaction of the Bioherbicide <i>Myrothecium verrucaria</i>with Technical-Grade Glyphosate on Glyphosate-Susceptible and -Resistant Palmer Amaranth

Previously we found that a strain of Myrothecium verrucaria (MV) exhibited bioherbicidal activity against several important weeds, and that some commercial formulations of glyphosate applied with MV resulted in synergistic interactions that improved weed control efficacy. We also found that MV had bioherbicidal activity against glyphosate-resistant Palmer amaranth. We have also reported that some commercial formulations are inhibitory to MV. Our objectives were to test the effect of unformulated glyphosate (high purity, technical-grade glyphosate) alone and in combination with MV for bioherbicidal activity on glyphosate-susceptible and -resistant Palmer amaranth biotypes under greenhouse conditions and to examine technical-grade glyphosate on the growth of this bioherbicide. High purity glyphosate (without adjuvants/surfactants) was not toxic to MV growth and sporulation at concentrations up to 2.0 mM when grown on agar supplemented with the herbicide. Both biotypes were injured by MV and MV plus glyphosate treatments as early as 19 h after application (3 h after a dew period of 16 h). These injury effects increased and were more evident through the 6-day time course, when after 120 h the MV plus glyphosate treatment had killed all glyphosate-susceptible and -resistant plants. The interaction of glyphosate plus MV was synergistic toward the control of Palmer amaranth. Data strongly suggest that the active ingredient is responsible for the synergy previously found when this bioherbicide was combined with some commercial formulations of glyphosate. Results demonstrated that MV can control both glyphosate-resistant and -susceptible Palmer amaranth seedlings and act synergistically with high-purity glyphosate to provide improved weed control.

Employing Canopy Hyperspectral Narrowband Data and Random Forest Algorithm to Differentiate Palmer Amaranth from Colored Cotton

Palmer amaranth (Amaranthus palmeri S. Wats.) invasion negatively impacts cotton (Gossypium hirsutum L.) production systems throughout the United States. The objective of this study was to evaluate canopy hyperspectral narrowband data as input into the random forest machine learning algorithm to distinguish Palmer amaranth from cotton. The study focused on differentiating the Palmer amaranth from cotton near-isogenic lines with bronze, green, and yellow leaves. A spectroradiometer was used to acquire hyperspectral reflectance measurements of Palmer amaranth and cotton canopies for two separate dates, December 12, 2016, and May 14, 2017. Data were collected from plants that were grown in a greenhouse. The spectral data were aggregated to twenty-four hyperspectral narrowbands proposed for study of vegetation and agriculture crops. Those bands were tested by the conditional inference version of random forest (cforest) to differentiate the Palmer amaranth from cotton. Classifications were binary: Palmer amaranth and cotton bronze, Palmer amaranth and cotton green, and Palmer amaranth and cotton yellow. Classification accuracies were verified with overall, user’s, and producer’s accuracy. For the two dates combined, overall accuracy ranged from 77.8% to 88.9%. The highest overall accuracies were observed for the Palmer amaranth versus the cotton yellow classification (88.9%, December 12, 2016;83.3%, May 14, 2017). Producer’s and user’s accuracies range was 66.7% to 94.4%. Errors were predominately attributed to cotton being misclassified as Palmer amaranth. The overall results indicated that cforest has moderate to strong potential for differentiating Palmer amaranth from cotton when it used hyperspectral narrowbands known to be useful for vegetation and agricultural surveys as input variables. This research further supports using hyperspectral narrowband data and cforest as decision support tools in cotton production systems.

Comparing Canopy Hyperspectral Reflectance Properties of <i>Palmer amaranth</i>to Okra and Super-Okra Leaf Cotton

Palmer amaranth (Amaranthus palmeri S. Wats.) is a major weed problem of cotton (Gossypium hirsutum L.) production systems in the southern United States. Hyperspectral remote sensing has shown promise as a tool for crop weed discrimination, and there is a growing interest in using this technology for identifying weeds in cotton production systems. Information is lacking on differentiating Palmer amaranth from cotton with an okra leaf structure based on canopy hyperspectral reflectance properties. Two greenhouse studies were conducted to compare canopy hyperspectral reflectance profiles of Palmer amaranth to canopy hyperspectral reflectance profiles of okra and super-okra leaf cotton and to identify optimal regions of the electromagnetic spectrum for their discrimination. Ground-based hyperspectral measurements of the plant canopies were obtained with a spectroradiometer (400 - 2350 nm range). Analysis of variance (ANOVA, p ≤ 0.05), Dunnett’s test (p ≤ 0.05), and difference and sensitivity measurements were tabulated to determine the optimal wavebands for Palmer amaranth and cotton discrimination. Results were inconsistent for Palmer amaranth and okra leaf cotton separation. Optimal wavebands for distinguishing Palmer amaranth from super-okra leaf cotton were observed in the shortwave infrared region (2000 nm and 2180 nm) of the optical spectrum. Ground-based and airborne sensors can be tuned into the shortwave infrared bands identified in this study, facilitating application of remote sensing technology for Palmer amaranth discrimination from super-okra leaf cotton and implementation of the technology as a decision support tool in cotton weed management programs.

Genomic Stability of Palmer amaranth Plants Derived by Macro-Vegetative Propagation

qPCR (quantitative polymerase chain reaction) and random amplified polymorphic DNA (RAPD) were utilized to investigate genetic stability of Palmer amaranth cloned plants over 10 generations. DNA from original parent Palmer amaranth plants (grown from seeds) was re-analyzed using qPCR, and confidence levels for determining ΔΔCt (threshold crossing) values were established. ANOVA was used to determine variation (margin of error) of these ΔΔCt values. This margin of error was applied to qPCR analysis of DNA from eight individual parent plants and their descendants (10th generation) so that possible differences in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene copy number could be ascertained. This method (and the associated error) indicated a lack of agreement in ΔΔCt values of DNA from plants of these two generations. qPCR analysis showed that in five out of eight clones, EPSPS gene copy number varied more than the calculated error (P = 0.05). A second technique to monitor genetic stability, RAPD was used to determine possible changes in genomic DNA due to extended cloning of these regenerated plants. RAPD analysis showed that four out of the eight clones differed when the profiles of the two generations were compared. Results show that qPCR and RAPD analysis point to the fact that several Palmer amaranth clones experienced changes in genome structure over 10 generations. Although the glyphosate resistance trait was retained, results suggest that during cloning studies, the genetic stability of macro-vegetatively propagated lines should be monitored.

Varying Tolerance to Glyphosate in a Population of Palmer Amaranth with Low EPSPS Gene Copy Number

A Palmer amaranth population (seeds collected in the year 2000;Washington Co., MS) suspected to be susceptible to glyphosate was examined as a population and as individual plants and found to exhibit varying tolerance or resistance to glyphosate. Whole plant spraying of glyphosate (0.84 kg·ha?1) to the population revealed that approximately 40% of this population were resistant to glyphosate and an LD50 of 0.75 kg·ha?1 was determined. Spray application of glyphosate indicated that some plants displayed varying degrees of resistance 14 days after treatment. Initial tests using leaf disc bioassays on 10 individual plants selected randomly from the population, allowed characterization of glyphosate resistance using both visual ratings of injury and quantitative measurement via chlorophyll content analysis. After initial bioassays and spray application, five plants with a range of tolerance to glyphosate were selected for cloning so that further studies could be accomplished on these individuals. Q-PCR analysis of these clones showed that resistance was not due to elevated EPSPS gene copy number. Shikimate levels were lower in the resistant and higher in the susceptible clones which correlated with varying degrees of resistance demonstrated in bioassays and spray application of glyphosate of these clones. Results demonstrate that individuals in a population can vary widely with respect to herbicide resistance and suggest that uptake, translocation, sequestration, metabolism or altered target site may contribute to the resistance in some individuals of this population.

一个抗咪唑乙烟酸长芒苋种群的发现

以咪唑乙烟酸为靶标除草剂,就几种苋属杂草对咪唑乙烟酸的抗药性进行研究。结果表明,供试皱果苋、反枝苋和其中的一个长芒苋P1种群(北京种群)对咪唑乙烟酸比较敏感,而在长芒苋中发现一个对咪唑乙烟酸有抗药性的P2种群(山东种群),在4倍推荐剂量下仍能全部存活。分子检测发现该种群ALS基因的第574位和第653位氨基酸发生了单碱基突变,分别为色氨酸(Trp)突变为亮氨酸(Leu)、丝氨酸(Ser)突变为天冬氨酸(Asp),种群中上述2种突变类型所占比例分别为38.9%、58.3%,2.8%的植株未检测到突变。

靶标基因突变导致入侵性杂草长芒苋对咪唑乙烟酸产生抗性

长芒苋Amaranthus palmeri生长迅速,适应性广,繁殖系数高,具有很强的竞争性,已在我国多地定植,对作物产量及生态环境构成潜在威胁。一旦其对除草剂产生抗性,将大大增加治理难度。本试验研究了采自不同地点的长芒苋种群对除草剂咪唑乙烟酸的抗性水平和抗性机理。整株生物测定得出,长芒苋疑似抗性种群和敏感种群对咪唑乙烟酸的GR_(50)分别为6584.18 g/hm^(2)和19.38 g/hm^(2),抗性指数(RI)高达339.78。对抗性突变位点的分子检测发现,疑似抗性种群部分植株ALS基因574位色氨酸突变为亮氨酸(Trp-574-Leu),部分植株653位丝氨酸突变为天冬氨酸(Ser-653-Asp),除此之外,35%的植株还发生了Trp-574-Leu和Ser-653-Asp双位点突变。表明我国已存在对咪唑乙烟酸产生高水平抗性的长芒苋种群,主要抗性机理为ALS靶标位点突变。本研究首次在我国发现了长芒苋种群对除草剂咪唑乙烟酸的靶标抗性。而入侵性杂草对除草剂产生抗性也对其治理提出了新的挑战。

影响长芒苋种子萌发因素的研究

长芒苋是我国1985年发现的外来入侵植物,明确长芒苋种子萌发特性,将为其有效防控提供数据支撑。采用室内生物测定方法,对温度、光照、水势、发芽深度等影响长芒苋萌发的关键因子进行研究。结果表明,变温处理可有效提高长芒苋种子的发芽势和萌发率,最适萌发温度为35℃/15℃变温处理;在35℃/15℃培养条件下,光照对长芒苋种子的萌发影响不显著;当渗透势为-0.4~-0.2 MPa时,长芒苋种子的萌发率可达82.2%~100%,当渗透势为-0.8 MPa时,仍有36.7%的种子萌发;长芒苋种子的出苗率受播种深度的影响,在播种深度为0.5~1.0 cm时,萌发率最高。由此可见,长芒苋萌发具有广泛的环境适生范围,这也可能是其在我国大面积入侵的生态学基础。

基于种子发芽率评估长芒苋在我国不同纬度地区的入侵风险

外来入侵植物长芒苋Amaranthus palmeri已经传入我国多个地区,定量评估其入侵风险对制定高效防控措施具有重要意义。外来植物入侵风险应该是由传入后能发芽并完成生活史的繁殖体如种子的量决定的,但已开展的适生区预测等风险评估通常忽略了该因素。基于此,本研究先通过同质园试验,比较了采自我国不同纬度的11个长芒苋种群与来自原产地美国种群的种子发芽率,分析了种群间发芽率的差异及其与纬度的相关性;然后,通过一年内连续多次在不同纬度地区的交互种植试验,分析了不同纬度种群间种子的发芽率和完成生活史的入侵窗口期差异,并判断是否发生了本地适应;最后,根据发芽率和入侵窗口期评估了长芒苋在我国不同纬度地区的入侵风险。同质园试验表明,种子发芽率与种群所处纬度显著正相关(P<0.05),发芽率随纬度升高而增高。交互种植试验表明,不同种群发芽率的差异是由于本地适应导致的,长芒苋在我国的入侵窗口期随纬度升高而缩短。基于不同种群种子发芽率和入侵窗口期的纬度差异,我们判断长芒苋在我国中低纬度至中高纬度区域内入侵风险较高,应该重点防控。长芒苋在高纬度和低纬度地区的入侵风险相对较低。但长芒苋种子萌发的本地适应可能会增加其在高、低纬度地区的入侵风险。因此,亟须加强对已传入种群的监测预警与早期防控力度,抑制其繁殖增长和进一步的扩散蔓延。

检疫性杂草——长芒苋传入我国的风险研究

从地理分布、潜在危害性、受害植物经济重要性、传入定殖扩散的可能性及危险性管理难度等方面,对长芒苋传入我国的风险进行研究,并提出加强检疫等措施。

苋属杂草种子形态学研究

苋属隶属苋科,含多种有害杂草,其中包含检疫性杂草异株苋亚属(Subgen.Acnida L.)。苋属种子常随粮谷贸易进入我国,口岸多有截获,种子小型,长1mm左右,形态相近,不易鉴别。本文在前人研究基础上,对21种苋属植物种子进行形态学研究,为口岸检疫鉴定工作提供技术支持。

重要检疫性杂草长芒苋的分子检测

为实现检疫性杂草长芒苋(Amaranthus palmeri)的有效检测,本研究基于苋属植物可溶性淀粉合成酶I基因(SSSI),建立了一种序列特异性PCR方法,可实现从众多苋属种子中检测出长芒苋。结果显示,该方法特异性强、简便快捷,可为口岸检疫鉴定工作提供有效的技术支持。

外来入侵植物长芒苋在中国不同地区的表型变异与环境适应性

长芒苋(Amaranthus palmeri)是近年来入侵我国的超级杂草,已在我国从南到北的多个点定殖,一旦大面积扩散势必会对农业生产和生物多样性保护等构成极大威胁。长芒苋在我国入侵点的纬度跨度大于其原产地北美洲的分布,探明其在我国不同纬度的地理种群的环境适应机制,对评估扩散潜能、建立早期监测预警等防控措施具有重要意义。本研究在同质种植园实验条件下,通过测定长芒苋不同纬度地理种群的生活史、形态及生物量等11个性状指标来分析其在我国的表型变异与环境适应性。结果表明,在同质园条件下,长芒苋不同地理种群间株高等7个表型性状指标差异不显著,而开花和发芽时间、花序长以及比叶鲜重4个指标具有显著差异(p<0.01),且它们的表型变异随年平均温度(纬度)线性增加或降低。开花时间随纬度的升高而缩短,发芽时间、花序长和比叶鲜重随纬度的升高而增加。基于长芒苋不同纬度地理种群间表型性状的异同,我们推断表型可塑性促进了其在我国不同地理环境中的定殖,表型变异(如高纬度种群开花时间提前)可促进其环境适应性进化,拓展其在我国的适生性分布区,增强其向原产地适应环境梯度之外地区入侵和扩散蔓延的潜力。